Hsa_circRNA_001676 accelerates the proliferation, migration and stemness in colorectal cancer through regulating miR-556-3p/G3BP2 axis

Circular RNAs (circRNAs) play key roles in colorectal cancer (CRC) progression, but little is known about the biological functions of hsa_circRNA_001676 in CRC. Therefore, we explored the potential role of hsa_circRNA_001676 in CRC development. RT-qPCR was performed to determine hsa_circRNA_001676, miR-556-3p and Ras-GTPase-activating SH3 domain-binding-proteins 2 (G3BP2) levels in CRC tissues. Meanwhile, to evaluate the roles of hsa_circRNA_001676, miR-556-3p and G3BP2 on CRC, functional analysis of cell proliferation, migration and stemness were then performed. Our results showed that compared to normal tissues, hsa_circRNA_001676 and G3BP2 level was elevated, but miR-556-3p level was reduced in CRC tissues. Additionally, luciferase reporter results showed that hsa_circRNA_001676 was shown to target miR-556-3p, and G3BP2 was targeted by miR-556-3p. Hsa_circRNA_001676 or G3BP2 overexpression promoted CRC cell proliferation and migration. Conversely, miR-556-3p overexpression suppressed CRC cell proliferation and migration. Moreover, deficiency of hsa_circRNA_001676 or G3BP2 repressed the CRC cell proliferation, migration and stemness. Meanwhile, hsa_circRNA_001676 deficiency obviously reduced tumor growth and stemness in a CRC mouse xenograft model. Furthermore, hsa_circRNA_001676 deficiency notably reduced G3BP2 level, but elevated miR-556-3p level in tumor tissues from tumor-bearing mice. Mechanistically, hsa_circRNA_001676 targeted miR-556-3p to increase G3BP2 level, contributing to the progression of CRC. Collectively, hsa_circRNA_001676 was able to accelerate proliferation, migration and stemness in CRC through regulating miR-556-3p/G3BP2 axis, suggesting that hsa_circRNA_001676 may become a potential therapeutic target in treating CRC.


Reverse transcription-quantitative PCR (RT-qPCR) assay
Total RNA from cells or tumor tissues were extracted using the Redzol reagent (SBS Genetech Co.,Ltd.).Next, the cDNA was synthesized using the Surescript™ First-Strand cDNA Synthesis Kit (iGeneBio).After that, qPCR was conducted with the SYBR Green qPCR Master Mix (None ROX) kit (Servicebio).U6 was used as an internal control for miR-556-3p, and GAPDH was used as an internal control for hsa_circRNA_001676, G3BP2, OCT4 and Nanog.The 2 −ΔΔCt method was used for determining relative gene levels.

Colony formation assay
HT29 and SW480 cells were loaded onto 12-well plates overnight at 37 °C.After indicated treatment, cells were then incubated for 1 week at 37 °C.Thereafter, cells were stained with 0.1% crystal violet solution for 30 min.Finally, the colonies were captured under a light microscope.

Wound healing assay
HT29 and SW480 cells were plated into 12-well plates overnight at 37 °C.The wounds were then created by using a sterilized pipet tip.After 0, 24 or 48 h of incubation, images were taken using a light microscope.

Immunohistochemical (IHC) and immunofluorescence (IF) staining assays
For IHC assay, sections were blocked in normal goal serum at room temperature for 20 min and then probed with anti-G3BP2 antibody (No. ab190011), anti-Oct-4 antibody (No. ab200834), anti-Nanog antibody (No. ab109250, Abcam) overnight at 4 °C.Next, sections were stained with HRP-labeled secondary antibody (No. Ab6721) for 20 min at room temperature.After visualizing with DAB solution, photographs were taken with a light microscope (OLYMPUS).
For IF assay, sections were probed with anti-CD133 antibody (No. ab19898, Abcam) and anti-CD44 antibody (No. ab238464, Abcam) overnight at 4 °C and then probed with the fluorescently-labeled secondary antibody (No. ab150077).Finally, images were taken with a fluorescence microscope (OLYMPUS).The Image Pro Plus software was used for semiquantitative analysis.

Animal study
All animal experiments were approved by the Animal Care and Use Committee of the Peking University Cancer Hospital (Inner Mongolia Campus)/Affiliated Cancer Hospital of Inner Mongolia Medical University, and conducted in accordance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals and carried out in accordance with the ARRIVE guidelines.A total of 20 BALB/c nude mice (18-20 g, Vital River) were grouped into three groups randomly: Control, NC and sh-hsa_circRNA_001676 groups.Each mouse was injected subcutaneously with untransfected (control group; 2 × 10 6 cells) or transfected HT29 cells (NC group and sh-hsa_circRNA_001676 group; 2 × 10 6 cells).Tumor growth was monitored at indicated times.The tumor volume (V) was calculated as: tumor length × tumor width 2 /2.After 4 weeks, the mice were sacrificed by cervical dislocation under anesthesia (1% isoflurane inhalation), and tumors were dissected out from the sacrificed mice.

Hematoxylin and eosin (H&E) staining assay
Paraffin-embedded tumor tissues were cut into 4-μm thick slices.After dewaxing and rehydration, sections were then subjected to H&E staining.Finally, images were observed under a light microscope.

Statistical analysis
Each experiment was independently repeated at least three times.GraphPad Prism 8 software was used for statistical analysis.The differences between two groups were evaluating using an unpaired Student t-test.Meanwhile, one-way analysis of variance (ANOVA) was used for multiple comparison.Data are shown as mean ± standard deviation.P < 0.05 indicates statistical significance.

Hsa_circRNA_001676 level was elevated in CRC tissues
By comparing circRNA levels in five pairs of noncancerous tissues and CRC tissues from CRC patients, a circRNA expression profile was downloaded from the GSE142837 dataset.The volcano plot disclosed that compared to the matched normal tissues, hsa_circRNA_001676 level was obviously elevated in CRC samples (Fig. S1), which was verified by the RT-qPCR results in Fig. 1A.Moreover, compared to NCM460 cells, higher level of hsa_cir-cRNA_001676 was detected in CRC cells (Fig. 1B).Meanwhile, among these five CRC cells, hsa_circRNA_001676 was expressed at the highest level in HT29 cells and expressed at the lowest level in SW480 cells (Fig. 1B).Thus, we reduced hsa_circRNA_001676 level in HT29 cells by using sh-Hsa_circRNA_001676, and overexpressed hsa_circRNA_001676 level in SW480 cells by using Hsa_circRNA_001676-OE (Fig. 1C and D).Significantly, hsa_circRNA_001676 expression was declined in HT29 cells transfected with sh-hsa_circRNA_001676 plasmids and increased in SW480 cells transfected with hsa_circRNA_001676-OE plasmids (Fig. 1C and D).

Hsa_circRNA_001676 enhanced CRC cell proliferation, migration and stemness
To explore the role of hsa_circRNA_001676 in CRC, CCK-8 and colony formation assays were conducted.Obviously, silenced hsa_circRNA_001676 strongly repressed HT29 cell viability and proliferation, whereas forced expression of hsa_circRNA_001676 enhanced SW480 cell viability and proliferation (Fig. 1E and F).Additionally, the results of transwell, wound healing and spheroid formation assays indicated that silenced hsa_cir-cRNA_001676 weakened HT29 cell migration and sphere formation abilities, whereas hsa_circRNA_001676 overexpression displayed the opposite effects on SW480 cells (Fig. 2A-C).Collectively, hsa_circRNA_001676 could accelerate CRC cell growth, migration and stemness.

G3BP2 is a downstream binding target of miR-556-3p
Next, Starbase (https:// starb ase.sysu.edu.cn/) database predicted the downstream targets of miR-556-3p.Over 3000 target genes were listed in Starbase.Among these, G3BP2 could regulate cancer stemness via upregulating the expressions of stem cell markers Oct-4 and Nanog 23 .Our results found that compared to normal controls, cell apoptosis was attenuated in CRC tissues (Fig. S2A).Meanwhile, the expressions of Oct-4, Nanog, CD133, CD44 and G3BP2 were obviously elevated in CRC tissues (Figs.S2B-E), suggesting that these tumor tissues displayed high self-renewal capability and high metastatic potential 28 .Meanwhile, the effect of G3BP2 on stemness in CRC and the relationship between G3BP2 and hsa_circRNA_001676/miR-556-3p in CRC remain elusive.Thus, we focused on G3BP2 because of its role in cancer stemness in the study.The data in Starbase showed that 3'UTR of G3BP2 was complementary to miR-556-3p sequences (Fig. 5A).Additionally, miR-556-3p mimics notably repressed the luciferase activity in wt-G3BP2 group cells (Fig. 5B), suggesting G3BP2 could be a direct target of miR-556-3p.
Furthermore, silenced G3BP2 greatly declined G3BP2 level in HT29 cells, as well as repressed HT29 cell viability, proliferation and migration; conversely, G3BP2 overexpression strongly elevated G3BP2 level in SW480 cells, as well as facilitated SW480 cell viability, proliferation and migration (Figs.5C-F and S3A-3B).Collectively, G3BP2 could act as an oncogene in CRC cells.

G3BP2 promoted CRC cell stemness
Next, we tested the effect of G3BP2 on CRC cell stemness in vitro.As revealed in Fig. 6A and B, Oct-4 and Nanog levels were declined in CRC cells by silencing of G3BP2, and increased by overexpression of G3BP2.Additionally, G3BP2 deficiency dramatically weakened the sphere formation ability of HT29 cells, whereas G3BP2 overexpression notably enhanced the sphere-forming capacity of SW480 cells (Fig. 6C).To sum up, G3BP2 could promote CRC cell stemness.

Discussion
CircRNAs have been shown to exhibit a regulatory role in CRC progression 29 .However, the biological role of a large number of circRNAs in CRC progression remain not well clarified.In this study, we found that hsa_circRNA_001676 level was elevated in CRC tissues and CRC cell lines.Additionally, downregulation of hsa_circRNA_001676 could suppress tumor growth and stemness in CRC in vitro and in vivo.Conversely, hsa_circRNA_001676 overexpression strongly accelerated tumor growth and stemness in CRC in vitro.Our results showed that hsa_circRNA_001676 could function as a new oncogenic driver to facilitate tumor growth and stemness in CRC.
Cancer stemness has been recognized as the leading cause of cancer metastasis and relapse 30 .Evidence has shown that some cancer cells possess typical stemness properties, which are important for the initiation and metastasis of tumors 31 .Targeting cancer stemness is a promising approach to fight CRC 32 .CircRNAs play a key role in modulating cancer stemness 33,34 .For example, CircPTN enhanced glioma cell stemness and self-renewal via sponging miR-145-5p 35 .CircAGFG1 could facilitate the stemness and metastasis in CRC through targeting miR-4262 and miR-185-5p 36 .Circ_0030586 retarded bladder cancer cell growth and stemness through targeting miR-665 37 .Our data showed that deficiency of hsa_circRNA_001676 remarkably repressed the sphere-formation ability of CRC cells in vitro and reduced CD133, CD44, Oct-4 and Nanog levels in tumor tissues in vivo, suggesting that deficiency of hsa_circRNA_001676 could suppress CRC cell stemness.However, the mechanism by which hsa_circRNA_001676 affects the stemness in CRC remains unknown.
CircRNAs have been reported to exert their regulatory roles in cancer development via functioning as miRNA "sponges" 38 .In this study, Circbank and CircInteractome databases were applied to predict the targets of hsa_cir-cRNA_001676.The data showed that hsa_circRNA_001676 could interact with miR-556-3p.Li et al. found that inhibition of miR-556-3p could enhance gastric cancer cell growth 21 .Conversely, diminished miR-556-3p repressed hemangioma cell proliferation through upregulating VEGFC 22 .These findings demonstrated that  www.nature.com/scientificreports/miR-556-3p may be expressed both as a tumor suppressor or an oncogene in different cancers.Our results showed that compared to normal colonic mucosa cells, miR-556-3p level was remarkably reduced in CRC cells.Forced miR-556-3p expression greatly repressed CRC cell proliferation and migration, suggesting that miR-556-3p might tumor suppressor gene in CRC.Furthermore, in this study, miR-556-3p was shown to target G3BP2.It has been shown that G3BP2 often overexpressed in some cancers and acted as an oncogene 39,40 .Overexpression of G3BP2 could expedite ESCC cell migration and invasion 39 .Inhibition of G3BP2 could decline PD-L1 expression in cancer cells, thereby facilitating anticancer immunotherapy 40 .Consistent with previous studies, we verified that G3BP2 overexpression could enhance CRC cell proliferation and migration, indicating that G3BP2 could act as an oncogene in CRC.Additionally, G3BP2 could trigger tumor initiation in breast cancer via upregulation of Oct-4 and Nanog 23 .Meanwhile, Oct-4 and Nanog are important stemness-associated mediators in the maintenance of cancer stemness 41,42 .Reducing Oct-4 and Nanog levels in CRC cells could repress the cancer stemness properties 43 .Moreover, high Oct-4 and Nanog levels are related to worse prognosis in CRC 44 .These findings above suggested a relationship between G3BP2 and cancer stemness.For the first time, we found that G3BP2 overexpression could upregulate Oct-4 and Nanog levels in CRC cells, suggesting that G3BP2 could facilitate CRC cell stemness.
Furthermore, circHERC4 could elevate E-cadherin protein level in CRC through inactivating miR-556-5p, thereby facilitating tumor migration and metastasis 45 .Circ_0020378 could facilitate osteosarcoma cell migration and growth via sponging miR-556-5p 46 .Meanwhile, circ-ABCB10 could affect cisplatin sensibility in lung cancer through binding with miR-556-3p 47 .Moreover, circFNDC3B could repress oncogene G3BP2 via binding with miR-1178-3p, thus suppressing bladder cancer progression 48 .These findings showed the relationships between different circRNAs and miR-556 or G3BP2.In this research, forced hsa_circRNA_001676 expression notably reduced miR-556-3p level, and elevated G3BP2, Oct-4 and Nanog levels in tumor tissues.These results showed that hsa_circRNA_001676 could act as a miR-556-3p sponge to weaken the inhibitory effects of miR-556-3p on its target G3BP2, thereby upregulating Oct-4 and Nanog levels.
It has been shown that one miRNA can target multiple genes and one circRNA also can sponge various miRNAs 49,50 .In this study, we only illustrated that hsa_circRNA_001676 could affect CRC cell growth and stemness through miR-556-3p/G3BP2 axis.However, further study is needed to uncover more hsa_ circRNA_001676-associated miRNAs or mRNAs, and this research might further illustrate the molecular mechanism of hsa_circRNA_001676 in CRC.

Conclusions
This study is the first to research hsa_circRNA_001676/miR-556-3p/G3BP2 axis in CRC.In summary, Hsa_ circRNA_001676 was able to accelerate proliferation, migration and stemness in CRC through targeting

Ethical approval and consent to participate Informed
consent was got from all participants and the Ethics Committee of Peking University Cancer Hospital (Inner Mongolia Campus)/Affiliated Cancer Hospital of Inner Mongolia Medical University approved this study.All animal experiments were approved by the Animal Care and Use Committee of the Peking University Cancer Hospital (Inner Mongolia Campus)/Affiliated Cancer Hospital of Inner Mongolia Medical University, and conducted in accordance with the National Institutes of Health Guidelines for Care and Use of Laboratory Animals and carried out in accordance with the ARRIVE guidelines.